The present invention relates to a method for extractive refolding of scrambled and polymerised single-chain polypeptides and proteins into their native conformation directly from a microbial fermentation broth.
Many polypeptides or proteins feature one or more intra molecular disulfide bonds serving to lock the tertiary conformation in place. If such polypeptides or proteins are recovered with incorrectly positioned disulfide bonds, they are often less biologically active than the corresponding molecule with correct disulfide bonds. Such scrambled or polymerised products are often found in proteins and polypeptides formed by fermentation of recombinant microorganisms or during the growth of a mammalian cell culture and may accumulate, stabilised by the incorrect disulfide bonds. The incorrect disulfide bonds may be intramolecular, giving rise to a misfolded monomeric product, or intermolecular, giving rise to dimeric or polymeric products.
The desired product and its accompanying, incorrectly formed, by-products may be secreted from the cells and found in soluble form in the broth. The may also be found in the periplasm as incorrectly folded and aggregated products resembling insoluble inclusion bodies. This will typically be the case when yeast is being used as the host organism for the recombinant production.
Alternatively, the desired product and its accompanying, incorrectly formed, by-products may be found either in the periplasm or inside the cells, in the form of insoluble inclusion bodies. This is the case when many polypeptides or proteins such as human proinsulin are expressed in E. coli 
Formation of such scrambled or polymerised by-products may be caused by a number of factors, e.g. that the conditions in the production host, such as pH, temperature, redox potential, concentration and type of chaperones etc. are different from those in the original (natural) site of production. Alternatively, the rate of production may be to high to cope with the time necessary for the molecule to fold into the native conformation which is usually believed to be the state having the lowest free energy. These scrambled and polymerised by-products represent a serious loss in commercial production. Therefore great efforts have been exerted to extract and refold these incorrectly folded proteins.
Thus, Steiner and Clark, Proc. Nat. Acad. Sci. 60, 622-629, 1968, disclose a method for isolation of reduced proinsulin and oxidation of the reduced form to native proinsulin; EP 600372 discloses a method for dissolving and extracting reduced proinsulin from E. coli inclusion bodies after cell homogenisation; EP 906918 discloses an improvement of this process by dilution of the reduced and extracted product in water; Frank, Pettee, Zimmerman and Burck, in: Peptides, Synthesisxe2x80x94Structurexe2x80x94Function, Proceedings of the Seventh American Peptide Symposium, Eds.: Rich, Gross, Pierce Chemical Company, Rockford, Ill., pp 729-738, 1981, describe a process in which human proinsulin is recovered from E. coli via its purified hexa S-sulfonate; WO 96/32407 discloses an unfolding and refolding process of secreted IGF-1; Markussen, in: Proinsulin, Insulin, C-Peptide, Proceedings of the Symposium on Proinsulin, Insulin and C-Peptide, Eds.: Baba, Kaneko, Yanaihara, Excerpta Medica, Amsterdam-Oxford, pp 50-61, 1979, discloses reduction of a single-chain des(B30) insulin precursor; Markusen, in: Int. J. Peptide Protein Res. 25, 431-434, 1985 compares refolding of a mini-proinsulin B(1-29)-A(1-21) and porcine insulin by air oxidation of the reduced and isolated forms of the molecules; and U.S. Pat. No. 6,003,875 discloses a method for improving the yield of IGF-1 when secreted from a yeast cell. A common feature with the prior art is that rather harsh conditions are used in the unfolding/refolding steps and that chaotropic agents are used requiring additional purification steps.
The present invention relates to a simple method for extractive refolding of scrambled and/or polymerised single-chain polypeptides or proteins to their native conformation directly from a microbial fermentation broth, in which they may appear in a variety of scrambled, misfolded and polymer forms together with the native, monomolecular form.
In the process according to the present invention, the sulphur chemistry is conducted directly on the crude product in the broth from the fermentation or, in the case the scrambled products are retained inside the cells, after disruption of the cell walls.
Thus, the present inventions is related to a method for extractive refolding scrambled and/or polymerised single-chain polypeptides contained in a microbial culture broth said method comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11;
b) adding a catalyst for refolding of disulfide bonds without adding a chaotropic agent;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) subjecting the supernatant to oxidation;
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
Steps a) to f) may be conducted in a temperature interval from about 4 to about 35xc2x0 C. or from about 20 to about 25xc2x0 C. and may be completed within a period from about 30 to about 180 minutes or from about 30 to 120 minutes. The order of one or more of steps a) to e) may be changed or reversed and some of the steps may even be omitted, e.g. step c). Thus in one embodiment, cells and cell debris are removed prior to step b). The catalyst for refolding disulfide bonds is typically a thiol compound, such as cysteine, HCl.
In one embodiment the present invention is related to a process comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11 by addition of diluted alkali hydroxide;
b) adding a thiol to the culture broth;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) stirring the supernatant under aeration;
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
In still another embodiment the present invention is related to a process comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11 by addition of 0.1-8 M sodium hydroxid;
b) adding solid or liquid cysteine, HCl to the culture broth to make 0.5-100 mM;
c) after a short period, e.g. 5 minutes, adjusting pH to about 7.0-11.0 if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) stirring the supernatant under aeration for a period of about 30 to about 180 minutes; and
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
In step a) the culture broth is advantageously diluted with water either before, simultaneously or after adjustment of pH. In one embodiment of the present invention, dilution of the culture broth is accomplished by addition of diluted alkali hydroxide. The dilution of the culture broth in step a) may be from about 2 to about 500%, from about 2 to about 300%, from about 2 to about 200%; from about 5 to 200%; from about 50 to 200% or from about 50 to 150%. Very high yields are obtained at a dilution of about 50%.
Thus in a further embodiment, the present invention is related to a method for extractive refolding scrambled and/or polymerised single-chain polypeptides or proteins contained in a microbial culture broth said method comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11;
a1) dilution of the culture broth to from about 2% to 500%;
b) adding a catalyst for refolding of disulfide bonds without adding a chaotropic agent;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) subjecting the supernatant to oxidation;
f) and isolating single-chain polypeptide material with correctly positioned disulfide bonds by suitable purification steps.
The refolded single-chain polypeptides or proteins from step e) will be subjected to suitable purification steps. Thus step f) may cover direct chromatography of the solution from step e) or precipitation by isoelectric precipitation, salting-out or by crystallisation.
The refolded polypeptide may be the desired end product or it may be an intermediate or precursor for the desired end product. The present invention includes a final step wherein the intermediates or precursors are converted into the desired end product by suitable means.
The process according to the present invention is particularly well suited for single chain human insulin precursors or human insulin analogue precursors which are expressed and secreted from yeast cells as described in further detail below. After completion of the unfolding/refolding process the single chain insulin precursors or single chain insulin analogue precursors are converted into insulin or an insulin analogue by suitable means such as in vitro conversion as disclosed in further details below.
Thus, in another embodiment, the present inventions is related to a method for extractive refolding scrambled and/or polymerised single-chain insulin precursors or insulin precursor analogues contained in a microbial culture broth said method comprising the following steps:
a) adjusting pH of the culture broth to approximately 10-11;
b) adding a catalyst for refolding of disulfide bonds without adding a chaotropic agent;
c) adjusting pH if necessary;
d) centrifugation of the culture broth to separate cells and cell debris;
e) subjecting the supernatant to oxidation;
f) isolating the single-chain insulin precursor or insulin precursor analogue with correctly positioned disulfide bonds by suitable purification steps;
g) and converting the insulin precursor or insulin precursor analogue into human insulin or a human insulin analogue by suitable enzymatic conversion steps.
Abbreviations and Nomenclature
As used herein the term xe2x80x9cthiol compoundsxe2x80x9d comprises compounds such as cysteine, mercaptoethanol, glutathione, dithiothreitol, or salts or mixtures thereof.
As used herein the term xe2x80x9csingle-chain polypeptides and proteinsxe2x80x9d is meant to comprise a single peptide strand constituted of codable amino acid residues. The single-chain polypeptides will contain at least 2 cysteine residues. Examples of single-chain polypeptides are human proinsulin and human proinsulin analogue precursors. Polypeptides and proteins produces by fermentation of transformed microorganisms containing inserted DNA coding for the desired polypeptide or protein or a precursor therefore are the primary targets.
As used herein the term xe2x80x9cscrambledxe2x80x9d is meant to comprise polypeptides containing disulfide bonds other than those found in the native polypeptide or protein when formed during the natural biosyntheses, inside or outside the native cells.
By xe2x80x9cmicrobial culture brothxe2x80x9d is meant the culture broth obtained after cultivation of the microorganism comprising DNA encoding the desired product and still containing cells and cell debris.
With xe2x80x9cextractive refoldingxe2x80x9d is meant a process where expressed polypeptides or proteins associated or bond to the cell wall and/or entrapped in the periplasmic space are released to the culture broth.
With the term xe2x80x9cchaotropic agentsxe2x80x9d is meant a compound that is capable of breaking hydrogen bonds in an aqueous solution such as urea, and guanidinium hydrochloride.
By xe2x80x9cconnecting peptidexe2x80x9d or xe2x80x9cC-peptidexe2x80x9d is meant the connection moiety xe2x80x9cCxe2x80x9d of the B-C-A polypeptide sequence of a single chain preproinsulin-like molecule. Specifically, in the natural insulin chain, the C-peptide connects position 30 of the B chain and position 1 of the A chain. A xe2x80x9cmini C-peptidexe2x80x9d or xe2x80x9cconnecting peptidexe2x80x9d such as those described herein, connect B29 or B30 to A1, and differ in sequence and length from that of the natural C-peptide.
With xe2x80x9cdesB30xe2x80x9d, xe2x80x9cBxe2x80x2xe2x80x9d or xe2x80x9cB(1-29)xe2x80x9d is meant a natural insulin B chain lacking the B30 amino acid residue, xe2x80x9cA(1-21)xe2x80x9d or xe2x80x9cAxe2x80x9d means the natural insulin A chain, xe2x80x9cB(1-29)-A(1-21), B28 Aspxe2x80x9d means a single-chain insulin precursor with aspartic acid at position 28 of the B-chain and no C-peptide (B29 is linked to A1). This insulin analogue is also called xe2x80x9cinsulin aspartxe2x80x9d. With xe2x80x9cBxe2x80x2Axe2x80x9d is meant a single chain insulin precursor with B(1-29) linked directly to the A chain of insulin.
By xe2x80x9cinsulin precursorxe2x80x9d is meant a single-chain polypeptide which by one or more subsequent chemical and/or enzymatic processes can be converted into human insulin.
By xe2x80x9cinsulin precursor analoguexe2x80x9d is meant an insulin precursor molecule having one or more mutations, substitutions, deletions and or additions of the A and/or B amino acid chains relative to the human insulin molecule. The insulin analogues are preferably such wherein one or more of the naturally occurring amino acid residues, preferably one, two, or three of them, have been substituted by another codable amino acid residue. In one embodiment, the instant invention comprises analogue molecules having position 28 of the B chain altered relative to the natural human insulin molecule. In this embodiment, position 28 is modified from the natural Pro residue to one of Asp, Glu, Lys, or Ile. In a preferred embodiment, the natural Pro residue at position B28 is modified to an Asp residue. In another embodiment Lys at position B29 is modified to Pro; Also, Asn at position A21 may be modified to Ala, Gln, Glu, Gly, His, Ile, Leu, Met, Ser, Thr, Trp, Tyr, Trp or Val, in particular to Gly, Ala, Ser, or Thr and preferably to Gly. Furthermore, Asn at position B3 may be modified to Lys. Further examples of insulin precursor analogues are des(B30) human insulin, insulin analogues wherein PheB1 has been deleted; insulin analogues wherein the A-chain and/or the B-chain have an N-terminal extension and insulin analogues wherein the A-chain and/or the B-chain have a C-terminal extension. Thus one or two Arg may be added to position B 1. Also, position 8 in the B chain may be modified to Asp or Trp and the position 11 in the B chain may be modified to Val.
The single-chain insulin precursors and insulin precursor analogues may be expressed with an N-terminal amino acid residue extension, as described in U.S. Pat. No. 5,395,922, and European Patent No. 765,395A. The N-terminal extension may be removed from the recovered refolded single-chain insulin precursor or insulin precursor analogue by means of a proteolytic enzyme which is specific for a basic amino acid (e.g. Lys) so that the terminal extension is cleaved off at the Lys residue. Examples of such proteolytic enzymes are trypsin or Achromobacter lyticus protease I.
After the single chain insulin precursor or insulin precursor analogue has been subjected to the defolding/refolding steps a)-f) it will be subjected to various in vitro procedures to remove the possible N-terminal extension sequence and to remove a possible connecting peptide or C-peptide to give insulin or the desired insulin analogue. Such methods include enzymatic conversion by means of trypsin or an Achromobacter lyticus protease in the presence of an L-threonine ester followed by conversion of the threonine ester of the insulin or insulin analogue into insulin or the insulin analogue by basic or acid hydrolysis as described in U.S. patent specification Ser. No. 4,343,898 or 4,916,212.
The single-chain insulin precursor or insulin precursor analogue may feature a peptide bridge linking residue Lys B29 to Gly A1, or they may feature a bridge or connecting peptide having from 1 to 36 amino acid residues. The insulin precursor or insulin precursor analogue may typically have a connecting peptide of from 1 to 5 or from 1 to 3 amino acids residues.
In another embodiment the single-chain protein or polypeptide may be IGF-I, hGH, or factor VII.
The pH value in step a) may be adjusted to about 10.5. Furthermore, the thiol added in step b) may be xcex2-mercaptoethanol, glutathione, dithiothreitol, or salts or mixtures hereof. The thiol may be added in an amount to make from 0.2 to 100 mM; from 1 to 100 mM; from 2 to 100 mM; from 2 to 50 mM; from 2 to 25 mM; from 2 to 15 mM; or from 2 to 5 mM in the culture broth. The thiol is typically added directly to the culture broth but may also be added after cells and cell debris have been removed e.g. by centrifugation. In another embodiment, the thiol is added after disruption of the cells, e.g. when the expressed single-chain polypeptide is not secreted from the cells.
In yet another embodiment the present invention relates to a method in which the temperature of step a) is adjusted from about 10 to 30xc2x0 C., and in a still further embodiment the temperature of step a) is from about 15 to about 25xc2x0 C.
In a further embodiment the present invention relates to a method in which step a) is completed in a time period from about 1 to about 20 minutes or from about 2 to about 15 minutes. In a further embodiment step a) is completed within about 10 minutes.
It may be necessary to adjust the pH after addition of the thiol in step b). Thus pH may be adjusted to about 8.5-10.2. In another embodiment pH may be adjusted to about 9.7.
The oxidation in step e) may be accomplished by adding air or oxygen to the supernatant from the previous step. Sufficient oxidation may also be accomplished without any special measurements. In the latter case oxidation is accomplished by aerating of the supernatant during stirring.
The method according to the present invention may be carried out in a scale from about 10 m3 to about 1000 m3. The method may be a continuous method with average holding times as described above for the batch operations.
The present invention is described in further detain in the following examples which are not in any way intended to limit the scope of the invention as claimed.